Genome-Wide Identification and Expression Analysis of Two-Component System Genes in Tomato

Abstract

The two-component system (TCS), which comprises histidine kinases (HKs), phosphotransfers (HPs), and response regulator proteins (RRs), plays pivotal roles in regulating plant growth, development, and responses to biotic and abiotic stresses. TCS genes have been comprehensively identified and investigated in various crops but poorly characterized in tomato. In this work, a total of 65 TCS genes consisting of 20 HK(L)s, six HPs, and 39 RRs were identified from tomato genome. The classification, gene structures, conserved domains, chromosome distribution, phylogenetic relationship, gene duplication events, and subcellular localization of the TCS gene family were predicted and analyzed in detail. The amino acid sequences of tomato TCS family members, except those of type-B RRs, are highly conserved. The gene duplication events of the TCS family mainly occurred in the RR family. Furthermore, the expansion of RRs was attributed to both segment and tandem duplication. The subcellular localizations of the selected green fluorescent protein (GFP) fusion proteins exhibited a diverse subcellular targeting, thereby confirming their predicted divergent functionality. The majority of TCS family members showed distinct organ- or development-specific expression patterns. In addition, most of TCS genes were induced by abiotic stresses and exogenous phytohormones. The full elucidation of TCS elements will be helpful for comprehensive analysis of the molecular biology and physiological role of the TCS superfamily.

Keywords: evolution; expression profiles; phylogeny; tomato; two-component system.

Figure 1

Phylogenetic analysis, gene structure, and conserved motifs of all HK(L) genes in tomato. (A) The phylogenetic tree of HK(L) proteins. Predicted amino acid sequences of HK(L) proteins were aligned using the Clustal X v1.81 program. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replicates as implemented in the MEGA 5.0; (B) Gene structure was analyzed using the Gene Structure Display Server online. The green boxes indicate the exons, and lines indicate the introns; (C) Schematic distribution of conserved motifs in the HK(L) proteins. Motif analysis was performed using MEME 4.0 software as described in the methods. The colored boxes represent different motifs in the corresponding position of each HK(L) protein.

Figure 2

Phylogenetic analysis (A); gene structure (B); and conserved motifs (C) of the HP family members in tomato. For other details, see Figure 1.

Figure 3

Phylogenetic analysis (A); gene structure (B); and conserved motifs (C) of RR genes in tomato. For other details, see Figure 1.

Figure 4

Phylogenetic relationship of HK(L) proteins in Arabidopsis, rice, maize, Chinese cabbage, soybean, Lotus japonicus, Physcomitrella patens, wheat, and tomato. The phylogenetic trees were constructed using the NJ method with bootstrap 1000 tests by MEGA 5.0. The diverse subgroups of HK(L) proteins were marked by different colors. The bar represents the relative divergence of the sequences examined.

Figure 5

Phylogenetic phylogenetic analysis of plant HP family genes in Arabidopsis, rice, maize, Chinese cabbage, soybean, Lotus japonicus, Physcomitrella patens, wheat, and tomato. For other details, see Figure 4.

Figure 6

Phylogenetic relationship of RR proteins in Arabidopsis, rice, maize, Chinese cabbage, soybean, Lotus japonicus, Physcomitrella patens, wheat, and tomato. For other details, see Figure 4.

Figure 7

Chromosomal distribution of TCS genes in tomato. The chromosome number is indicated at the top of each chromosome. The arrows indicate the sense (▲) and antisense (▼) strands. The pairs of genes with tandem duplication were highlighted with the yellow background. The duplicated gene pairs have been link by dark line.

Figure 8

Subcellular localization of TCS proteins. Green fluorescent protein (GFP)-fusion proteins were transiently expressed in onion epidermis cells under the control of the 35S promoter. After 16–18 h of incubation, GFP signal was detected with a green fluorescence microscope. Fluorescence (up) and bright-field images (down) of plasmolyzed empty vector pFGC: GFP transgenic cell (A); Fluorescence (up) and bright-field images (down) of 35S::SlHK8-GFP (B); 35S::SlHP2-GFP (C); 35S::SlHP3-GFP (D); 35S::SlRR1-GFP (E); and 35S::SlRR8-GFP (F) transgenic cell. Scale bar was presented in bottom right.

Figure 9

Heat map representation for organ-specific expression (A) and six fruit development stages-related expression (B) profiles of TCS genes in tomato. These electronic expression data were downloaded from the tomato eFP browser at bar.utoronto.ca. The heatmap was drawn by MeV4.8. The expression levels are presented using fold-change values transformed to Log₂ format compared with control. The Log₂ (fold-change values) and the color scale are shown at the top of heat map. Green, black, and red represent low, medium, and strong expression, respectively.

Figure 10

The Gene Ontology (GO) analysis of TCS genes. The TCS genes were categorized into three groups: molecular function (A); biological process (B); and cell component (C).

Figure 11

Heat map representation for the response patterns to exogenous trans-zeatin (ZT) (A) and ABA (B) of TCS genes in tomato. The second true leaves were collected at 0, 1, 2, 4, and 8 h after 100 μM ZT or 100 μM ABA treatment. The heatmap were manufactured by MeV4.8. The color scale representing the relative expression values is shown in the upper left of the heatmap.

Figure 12

Heat map representation for the response patterns to drought (A) and salt (B) stresses of TCS genes in tomato. The second true leaves were collected at 0, 1, 2, 4, and 8 h after the onset of stress treatments. For other details, see Figure 10.